The goal of this proposal is to elucidate the molecular mechanisms that regulate intestinal permeability. Such regulation occurs in vivo as a consequence of transcellular Na+-nutrient transport and may result in paracellular amplification of nutrient absorption. Thus, characterization of the underlying mechanisms will enhance understanding of nutrient absroption and physiologic regulation of intestinal permeability. At the cellular level, the tight junction is the major determinant of paracellular permeability. Both structural and functional tight junction modifications accompany Na+-nutrient, e.g. Na+-glucose, cotransport in intact mucosa. However, the complexities of intact mucosa have limited further analysis of this process. In order to evaluate tight junction regulation in detail I have established a cell culture model of regulated paracellular (tight junction) permeability as a consequence of Na+-glucose cotransport. This proposal will test the hypothesis that Na+-nutrient cotransport initiates a signalling cascade which leads to structural and functional tight junction modifications resulting in augmented paracellular permeability. The proposed signalling pathway leading from Na+-glucose cotransport to altered tight junction permeability will be dissected into a series of sequential events. Additionally, the specific morphologic and biochemical alterations of the tight junction and tight junction-associated proteins that occur as a function of Na+-glucose cotransport will be evaluated. Experimental design will include functional, morhpologic, biochemical, and pharmacologic assessment of tight junctions during Na+-glucose cotransport.